Viruses within the Flavivirus genus, such as Japanese encephalitis (JE), St. Louis encephalitis (SLE), West Nile (WN), and tick-borne encephalitis (TBEV) viruses, are important human pathogens, typically causing a devastating and often fatal neurological disease. They represent a serious public health problem in many regions of the world. Due to the changes in climate and an increase in human population and travel, both mosquito- and tick-borne flaviviruses expanded their geographic range during the past decades and emerged in areas where they previously did not exist. The spread of viruses in new areas resulted in severe impact on wildlife and caused outbreaks of diseases in humans. Currently, WN is endemic throughout the North America and it is the major source of viral encephalitis in the USA. During the 1999-2014 outbreaks, nearly 3 million people were infected with WN in the US, with about 18,810 reported cases of encephalitis/meningitis that resulted in 1765 deaths, majority of which occurred in elderly. No licensed human vaccine is available to prevent WN disease. Also, despite the use of formalin-inactivated TBEV vaccines, the incidence of tick-borne encephalitis has increased in endemic areas of Europe and Asia during the past 2 decades. This increase in number of flavivirus associated illness emphasizes the need for effective vaccines that will induce a durable immunity and protection. The pathogenesis of neurotropic flavivirus infections involves two distinct properties of the viruses: neuroinvasiveness and neurovirulence. In the brain, the primary targets of neurotropic flaviviruses are neurons. Thus, successful attenuation of a neurotropic virus depends on the prevention of virus entry into the central nervous system (CNS) and restriction of its replication in the neurons. In order to limit virus access into the CNS, we utilized an approach in which non-neuroinvasive dengue flavivirus (DEN4) was used as a genetic background to create a new antigenic chimeric flavivirus containing structural protein genes derived from a wild-type neurotropic flavivirus (TBEV, JE, SLE, or WN). Chimerization reduced neuroinvasiveness, the ability of virus to replicate in the periphery and spread into the CNS, but viruses retained a high level of neurovirulence in mice and monkeys. In studies performed in our section in FY2013-14, we have proved that an approach based on targeting of the flavivirus genome for number of cellular microRNAs (miRNAs) abundantly expressed in the brain is a simple and efficient method to control the replication and pathogenesis of neurotropic flavivirus (chimeric tick-borne encephalitis virus/dengue type 4 virus; TBEV/DEN4) in a cell- or tissue-specific manner. Moreover, we found that simultaneous multiple miRNA co-targeting of two distantly located regions, the 3-noncoding region (3NCR) and the open reading frame (ORF), completely abolished the virus neurotropism as no viral replication was detected in the developing CNS of neonatal mice. Additionally, no viral antigens were detected in neurons, and neuronal integrity in the brain of mice was well preserved. In FY2015, immunogenic properties of these viruses were tested in rhesus monkeys, in which parental TBEV/DEN4 virus caused a high level, long-lasting viremia while the introduction of miRNA targets in the TBEV/DEN4 genome greatly attenuated the virus for monkeys; their mean peak viremia was reduced by a 50-fold. However, immunization with a single dose of these viruses induced robust TBEV-specific antibody response that was comparable to that of monkeys that received 3 human doses of commercially available inactivated TBEV vaccine. As a result, monkeys immunized with miRNA viruses were completely protected against challenge with neurovirulent TBEV/LGT virus. In 2015, principles of miRNA-targeting to control the virus replication in the CNS were applied to chimeric SLE/DEN4 and WN/DEN4 viruses. We demonstrated that insertion of targets for mir-9 and mir-124 miRNAs in the E protein gene and in three sites in the 3NCR of SLE/DEN4 or WN/DEN4 was sufficient to selectively inhibit viral replication in neurons and to constrain the development of lethal encephalitis in adult and newborn mice after intracerebral or intraperitoneal infection. Currently, evaluation of protective properties of a number of miRNA-targeted SLE/DEN4 and WN/DEN4 viruses in mice are underway. Flaviviruses are transmitted in nature by their specific arthropod vector, and the enviromental safety of live attenuated flavivirus vaccines is a significant concern. Hypothetically, the DEN4-based vaccine viruses (TBEV/DEN4, SLE/DEN4, and WN/DEN4 miRNA-targeted viruses) have a potential for reversion back to wild-type that could be associated with possible dissemination of these mutated viruses by mosquitoes after feeding on vaccinees. To mitigate this risk we developed a microRNA-targeting approach that selectively restricts replication of flavivirus in the mosquito host. Introduction of sequences complementary to a mosquito-specific mir-184 and mir-275 miRNAs individually or in combination into the 3NCR and/or ORF region resulted in selective restriction of DEN4 replication in mosquito cell lines and two species of Aedes mosquitoes, which are the widely distributed competent vectors for many arboviruses. We believe that the developed miRNA co-targeting approach can be adapted to support the design of environmentally safe, live attenuated virus vaccines by restricting their ability to be introduced into nature and limiting the possibility of subsequent viral evolution, leading to unpredictable consequences. In FY2015, we investigated the suitability of the miRNA-targeting approach for attenuation of the more neurovirulent chimeric virus (TBEV/LGT), which is based on the genetic backbone of the naturally attenuated, tick-borne Langat virus (LGT). Since LGT is antigenically closely related to TBEV strains, we assumed that LGT-based chimeric TBEV/LGT vaccines might provide greater humoral and cellular immunity against TBEV than our lead candidate based on the DEN4 genetic backbone. Unlike the TBEV/DEN4, the TBEV/LGT virus retained ability of its parental viruses to spread from peripheral site of inoculation to the CNS. We evaluated ten potential sites in the 3NCR of the TBEV/LGT genome for placement of microRNA targets and found that the TBEV/LGT genome is more restrictive for such genetic manipulations compared to that of TBEV/DEN4. In addition, unlike TBEV/DEN4 virus, the introduction of the multiple miRNA targets into either 3NCR or ORF of TBEV/LGT genome had only modest effect on virus attenuation in the developing CNS of highly permissive newborn mice. However, simultaneous miRNA-targeting in the ORF and 3NCR had more than additive effect on control and silencing of virus replication in the brain and completely abolished the virus neurotropism. Furthermore, neuroinvasiveness of miRNA-targeted TBEV/LGT viruses in very sensitive immunodeficient SCID mice was significantly limited but was not complete. In the absence of potent B and T cell responses prolonged replication of viruses in SCID mice was accompanied by emergence of escape mutants with large deletions of miRNA-targets inserted in both sites of the genome leading to neurological disease. It appears that to provide more effective protection against neuroinvasive TBEV/LGT virus, the copy number of miRNA targets should be increased in many different genome regions. We are currently investigating possibility of the placement of additional miRNA targets in viral genome with the aim of tuning viral replication in the CNS and periphery.